Development of new eco-friendly supports for immobilization of enzymes based on cellulose residues

The pulp and paper industry generates a high volume of solid wastes that are usually burned to obtain energy, directed to landfills, or incinerated. Among the wastes generated in this process is the paper sludge, a residue rich in cellulose with low lignin content making it a useful raw material to produce high-value products such as cheap immobilization supports. In the current work, paper sludge was activated using different functional groups (amino, epoxy, and aldehyde). The xylanase GH10 from Malbranchea pulchella was used as a model enzyme for the immobilization assays. The enzyme was efficiently immobilized through reversible immobilization on aminated support monoaminoethyl-N-ethyl (MANAE) and polyethyleneimine (PEI), achieving yields of more than 90 %. Furthermore, the yield and activity of the biocatalyst immobilized with paper sludge using groups glyoxyl and epoxy (irreversible immobilization) were higher than the enzyme immobilized on agarose supports. The biocatalyst immobilized on paper sludge-epoxy presented the best results, reaching 12.54 U.g-1 of support. Therefore, the use of paper sludge, such as backbone of different immobilization supports, was an efficient method and promising approach for the immobilization of enzymes such as xylanase. More studies are necessary to optimize the displayed potential for future applications as tests in other enzymes of different characteristics and their behavior with bifunctional reagents as the glutaraldehyde. Furthermore, the valorization of these residues in a biorefinery context holds great socio-economical relevance for Portugal and Brazil. Thus, our perspectives are the development of a hybrid biocatalyst using magnetics nanoparticles and paper sludge.info:eu-repo/semantics/publishedVersio

Immobilization and stabilization of the endo-1,4-beta-xylanase of Malbranchea pulchella for production of the xylooligosaccharides

Over the last few years, the lignocellulosic biorefinery concept has been extended beyond the application of biofuel production. Innovative and efficient technologies for lignin, cellulose, and hemicellulose fractionation allow the implementation of integrated processes for the co-production of bioenergy and higher value-added bioproducts. Among the different approaches, the use of Endo-1,4--xylanases (EC 3.2.1.8) in the hydrolysis of rich-xylan feedstocks has increased in the integrated process to produce fermentable and xylooligosaccharides (XOS). Nowadays, XOS has been preferentially used as prebiotic components in the development of new functional foods for presenting additional biological benefits such as antioxidant, inflammatory, and immunomodulatory activities. In the current work, we immobilized an endo-1,4-beta-xylanase of Malbranchea pulchella (Mpxyn10) and evaluated its potential in the production of XOS from xylan from various sources. Mpxyn10 was immobilized on agarose-activated supports (Glyoxyl-, MANAE-, GLUT- and PEI-agarose) and commercial Purolite support. Values >90% of immobilization yield were obtained on aminoactivated supports (Purolite, MANAE, and PEI-agarose) after 120 min, and the highest values of activity recovery were obtained for MANAE-MpXyn10 (137%) and Purolite-MpXyn10 (142%) derivatives. MANAE- and Purolite-MpXyn10 derivatives maintained more than 90% of their activity after 24 h of incubation at 70 °C, while the residual activity of free MpXyn10 was only 11%. MpXyn10 derivatives were also active and stable over a wide range of pH (4.0-6.0) and in the presence of furfural and HMF compounds. MpXyn10 derivatives were tested to produce XOS from xylan from various sources. Maximum values of XOS (xylobiose and xylotriose) were found for xylan beechwood at 8.1 mg mL-1, birchwood at 8.6 mg mL-1, and wheat arabinoxylan at 8.9 mg mL-1 after 3 h of reaction, at 50 °C, using Purolite-MpXyn10. This derivative was reused in various reaction cycles, maintaining more than 80% of yield XOS after 6 cycles of reaction. The results obtained in this work provide a basis for the development of applications of immobilized MpXyn10 to XOS production and other high value-added product in the lignocellulosic biorefinery field.The work was supported by the following: FAPESP (São Paulo Research Foundation, grants: 2014/50884 and 2018/07522-6; Process 2020/00081-4) and National Institute of Science and Technology of Bioethanol, INCT, CNPq (grant: 465319/2014-9) and Process 301963/2017-7.info:eu-repo/semantics/publishedVersio

Comparison in the Trichoderma longibrachiatum xyloglucanase production using tamarind (Tamarindus indica) and jatobá (Hymenaea courbaril) seeds: factorial design and immobilization on ionic supports

in the control of the stretching and expansion of the plant cell wall. There are five types of enzymes known to be capable of cleaving the linear chain of xyloglucan, the most famous of them being the xyloglucanase (XEG). The immobilization can be used to solve problems related to stability, besides the economic benefits brought by the possibility of repeated use and recovery, decreasing the costs of production. Therefore, this study aims the optimization of the production of a xyloglucanase from Trichoderma longibrachiatum, with the aid of factorial design, using tamarind (Tamarindus indica) and jatobá (Hymenaea courbaril) seeds as carbon source; and the immobilization of the enzyme on ionic supports, such as MANAE (monoamino-N-aminoethyl), DEAE (diethylaminoethyl)-cellulose, CM (carboxymethyl)-cellulose and PEI (polyethyleneimine). High concentrations of carbon source in the culture medium, especially tamarind seeds, were the most favorable conditions for the greater activity of the xyloglucanase from T. longibrachiatum. The scaling up from Erlenmeyer flasks to the bioreactor was an essential strategy to increase the content of secreted enzyme. Regarding the biochemical characterization of the crude extract, the optimal temperature was 50-55 °C and the optimal pH 5.0. Regarding the stabilities to pH and to temperature, the enzyme was not stable for prolonged periods, which was crucial for the performing of immobilization on ionic resins (CM-cellulose, DEAE-cellulose, MANAE, and PEI), being the first time described in literature the immobilization of a xyloglucanase on these supports.We thank the Fundação de Amparo à Pesquisa do estado de São Paulo (process 2018/07522-6; 2014/50884-5), and Conselho Nacional de Dsenvolvimento Científico (process 301963/2017-7; 465319/2014-9).info:eu-repo/semantics/publishedVersio

Acompanhamento da construçao de obras complementares, integradas no aproveitamento hidroelétrico do baixo sabor

Trabalho final de mestrado para obtenção do grau de mestre em Engenharia Civil na área de Especialização em EdificaçõesO presente relatório de estágio enquadra-se no âmbito do Trabalho Final de Mestrado, do curso de Engenharia Civil, Área de Especialização de Edificações, ministrado no Instituto Superior de Engenharia de Lisboa (ISEL). O Estágio foi realizado entre meados de junho e dezembro 2014, na empreitada de construção do Empreendimento do Aproveitamento Hidroelétrico do Baixo Sabor (AHBS), empreitada adjudicada às empresas Bento Pedroso Construções, SA e Lena – Engenharia e Construções, SA, pela empresa EDP – Gestão da Produção de Energia, SA. Concretamente, o estágio traduziu-se no acompanhamento de processos construtivos no Empreendimento, em particular nas Obras Complementares, onde se destaca a Construção do novo Santuário de Santo Antão, com diversas edificações de apoio, 3 Restabelecimentos a Estradas Nacionais, 1 Restabelecimento a um caminho agrícola e 1 Restabelecimento a um caminho florestal. O estágio na empreitada, baseou-se nas seguintes atividades: • Planear, organizar e dirigir os trabalhos, garantindo a sua execução dentro dos prazos e orçamento; • Gerir as relações com os subempreiteiros, supervisionando os trabalhos; • Gerir equipas de trabalho do ACE, materiais e equipamentos envolvidos nas diversas obras. Quando se iniciou o estágio, a Empreitada já se encontrava na sua fase final.The present report refers to the Master’s Disserttion in the Civil Engineering– Buildings Specialization Area, held at the Lisbon’s Politechnic Engineering Institute (ISEL). The post-graduate training was performed between July and December 2014 within the construction contract of the Baixo Sabor Hydroelectric Project (AHBS), a contract awarded to the companies Bento Pedroso Construções, SA and Lena - Engenharia e Construções, SA, by EDP Gestão da Produção de Energia, SA. Objectively, the post-graduate training tasks were the follow up of construction processes in this Contract, in particular in the Complementary tasks, amongst them, being the new Santo Antão Sanctuary an highlight. Additionally, several supporting buildings, as well as 3 restorations of National Roads, 1 restoration of an agricultural path and 1 restoration of a forest path. The post-graduate training was based on the following activities: • Plan, organize and direct the tasks, ensuring its execution within the deadlines and budget; • Manage relations with subcontractors, supervise the subtasks; • Manage the work teams, materials and equipment involved in the various assignments. When the post-graduate training begins, the Contract is already in its final phase.N/

Beauveria bassiana Lipase A expressed in Komagataella (Pichia) pastoris with potential for biodiesel catalysis

et al.Lipases (EC 3.1.1.3) comprise a biotechnologically important group of enzymes because they are able to catalyze both hydrolysis and synthesis reactions, depending on the amount of water in the system. One of the most interesting applications of lipase is in the biofuel industry for biodiesel production by oil and ethanol (or methanol) transesterification. Entomopathogenic fungi, which are potential source of lipases, are still poorly explored in biotechnological processes. The present work reports the heterologous expression and biochemical characterization of a novel Beauveria bassiana lipase with potential for biodiesel production. The His-tagged B. bassiana lipase A (BbLA) was produced in Komagataella pastoris in buffered methanol medium (BMM) induced with 1% methanol at 30°C. Purified BbLA was activated with 0.05% Triton X-100 and presented optimum activity at pH 6.0 and 50°C. N-glycosylation of the recombinant BbLA accounts for 31.5% of its molecular weight. Circular dichroism and molecular modeling confirmed a structure composed of α-helix and β-sheet, similar to α/β hydrolases. Immobilized BbLA was able to promote transesterification reactions in fish oil, demonstrating potential for biodiesel production. BbLA was successfully produced in K. pastoris and shows potential use for biodiesel production by the ethanolysis reaction.This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo - Consejo Superior de Investigaciones Cientificas (FAPESP-CSIC, process 2013/50892-5), Conselho de Desenvolvimento Científico e Tecnológico (CNPq), National System for Research on Biodiversity (SisbiotaBrazil, CNPq 563260/2010-6/FAPESP n◦ 2010/52322-3) and CNPq Biodiesel Process n◦ 406838/2013-5. FAG Torres, JJ, MLTP and RJ Ward are Research Fellows of CNPq. FA Facchini was FAPESP fellows; AV and MGP were CNPq fellows, AC and CC were CAPES fellows.Peer Reviewe

Stabilization of the lipase of Hypocrea pseudokoningii by multipoint covalent immobilization after chemical modification and application of the biocatalyst in oil hydrolysis

Lipase from Hypocrea pseudokoningii was purified using the support Octyl-Sepharose. This adsorption resulted in a 3-fold increase in activity of the immobilized enzyme. Following, still on this support, the lipase was enriched in surface amino groups (by reaction of carboxy groups with ethylendiamine). After amination, the lipase was desorbed from Octyl-Sepharose, while the 2-fold hyper-activation was maintained. The aminated lipase was also successfully immobilized on Glyoxyl-Agarose. The derivative was 45-fold more stable than was the free enzyme at 50 and 60 °C. The derivative was also stable in 50% of organic solvents such as methanol, ethanol, propanol and cyclohexane. The multipoint immobilization also increased the enzyme stability in relation to the free enzyme in the presence of ethanol, methanol and cyclohexane for up to 72 h. For example, the stabilized derivative was 9-fold more stable than the free enzyme in presence of methanol. The derivatives hydrolyzed fish, cupuaçu (Theobroma grandiflorum), bacuri (Latonia insignis) and murumuru (Astrocaryum murumuru) oils. The multipoint immobilization process increased the hydrolysis of oils up to 15-fold compared with the control, what makes these derivatives attractive for industrial application.This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho de Desenvolvimento Científico e Tecnológico (CNPq process n° 406838/2013-5). This project is also part of the National Institute of Science and Technology of the Bioethanol (n° 574002/2008-1), CNPq—Ciência sem Fronteira (n° 242775/2012-8). JAJ and MLTMP are Research Fellows of CNPq. MGP and ACV are supported by CNPq. FDAF was recipient of a FAPESP Fellowship.Peer Reviewe

Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry

Hypocrea pseudokoningii purified lipase was immobilized on hydrophobic supports (phenyl-sepharose, butyl-sepharose, octyl-sepharose, Hexyl Toyopearl, Lewatit, Purolite, Decaoctyl sepabeads) and ionic supports (Duolite, DEAE-agarose, PEI-agarose, MANAE-agarose, and Q-sepharose). The immobilization processes resulted in derivatives with excellent thermal stabilities, increasing the half-life up to 500-fold. The derivatives had excellent stability to organic solvents compared to the crude lipase. In the presence of 50% ethanol, hexyl and Decaoctyl derivatives increased by about 6- and 3.5-fold their stability to organic solvents, respectively. When tested for methanol, phenyl-sepharose derivative also increased their stability to organic solvents in approximately 2-fold. Octyl-sepharose derivative was fully stable for 48 h in the presence of propanol, which showed a half-life of about 7.5 h. The greater activation of the derivatives occurred using 50% cyclohexane, in which the hexyl derivative obtained an increase in the activity of 9-fold and phenyl and octyl derivatives had their activity increased by 6-fold. The lipase showed activity on different oils. Therefore, the adsorption of lipases in low ionic strength and highly hydrophobic supports is shown to be a simple and rapid tool for the immobilization of H. pseudokoningii lipase. These derivatives strongly increase the chances of this biocatalyst for industrial application.This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho de Desenvolvimento Científico e Tecnológico (CNPq process n° 406838/2013-5). This project is also part of National Institute of Science and Technology of the Bioethanol (n° 574002/2008-1), CNPq – Ciência sem Fronteira (n° 242775/2012-8). JAJ and MLTMP are Research Fellows of CNPq. MGP and ACV are supported by CNPq. FDAF was recipient FAPESP Fellowship.Peer Reviewe

Trametes versicolor laccase production using agricultural wastes: a comparative study in Erlenmeyer flasks, bioreactor and tray

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00449-019-02245-z) contains supplementary material, which is available to authorized users.Laccases are very interesting biocatalysts of recognized importance for several industrial applications. Its production by Trametes versicolor, a white-rot fungus, was induced by a combination of cotton gin wastes (1\%), a lignocellulosic waste, and vinasse (15\%), an industrial by-product from sugarcane industry. The use of these agro-industrial wastes are interesting, since it helps in reducing the enzyme production costs, due to their low cost and wide availability, as well as the environmental contamination issues, due to their improper disposal. Thus, laccase production was studied in submerged fermentation of T. versicolor using these agro-industrial wastes (cotton gin waste and vinasse) as carbon source and an additional nitrogen source (0.1\\% peptone). Three different bioreactors were evaluated for laccase production, such as BioFlo 310 bioreactor, aluminium tray and Erlenmeyer flasks to achieve high levels of laccase production. The highest specific production of laccase was found in BioFlo 310 bioreactor with 12 days of fermentation (55.24 U/mg prot.), which has been shown to be closely related to the oxygen supply to the microorganism through aeration of the fermentation medium. This study brings new insights into green biotechnology regarding vinasse utilization, which is frequently discharged in soils, rivers, and lakes causing adverse effects on agricultural soils and biota, as well as the cotton gin waste recovery.The authors are grateful to Mariana Cereia and Maurício de Oliveira for their technical assistance. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, processes 2010/52322-3; 2014/50884-5; 2018/07522-6), and V. E. P. was the recipient of a FAPESP fellowship (Process 2015/23200-0).info:eu-repo/semantics/publishedVersio

Matrix Discriminant Analysis Evidenced Surface-Lithium as an Important Factor to Increase the Hydrolytic Saccharification of Sugarcane Bagasse

Statistical evidence pointing to the very soft change in the ionic composition on the surface of the sugar cane bagasse is crucial to improve yields of sugars by hydrolytic saccharification. Removal of Li+ by pretreatments exposing -OH sites was the most important factor related to the increase of saccharification yields using enzyme cocktails. Steam Explosion and Microwave:H2SO4 pretreatments produced unrelated structural changes, but similar ionic distribution patterns. Both increased the saccharification yield 1.74-fold. NaOH produced structural changes related to Steam Explosion, but released surface-bounded Li+ obtaining 2.04-fold more reducing sugars than the control. In turn, the higher amounts in relative concentration and periodic structures of Li+ on the surface observed in the control or after the pretreatment with Ethanol:DMSO:Ammonium Oxalate, blocked -OH and O− available for ionic sputtering. These changes correlated to 1.90-fold decrease in saccharification yields. Li+ was an activator in solution, but its presence and distribution pattern on the substrate was prejudicial to the saccharification. Apparently, it acts as a phase-dependent modulator of enzyme activity. Therefore, no correlations were found between structural changes and the efficiency of the enzymatic cocktail used. However, there were correlations between the Li+ distribution patterns and the enzymatic activities that should to be shown

Immobilization effects on the catalytic properties of two Fusarium verticillioides lipases: Stability, hydrolysis, transesterification and enantioselectivity improvement

This article belongs to the Special Issue Immobilized Biocatalysts.Fusarium verticillioides lipases were purified in a “cascade” method using octadecyl Sepabeads and octyl Sepharose resins, which led to the isolation of two proteins with lipolytic activities. Lip 1 was purified after octyl Sepharose adsorption presenting 30.3 kDa and, Lip 2 presented 68.0 kDa after octadecyl adsorption. These immobilization processes resulted in an increase of 3-fold in activity of each immobilized enzyme. These enzymes presented optima of pH of 5.0 and 6.0, respectively and temperature at 40 °C. They were thermostable at 40 °C and both remained more than 50% of its activity at the pH range of 5.0 to 7.0, with 180 min of incubation. The sardine oil hydrolysis showed higher EPA/DHA ratio. Concerning the ethanolysis reaction, Lip 2 showed higher conversion (5.5%) and both lipases showed activity in the release of the S enantiomers from 2-O-butyryl-2-phenylacetic acid (mandelic butyrate acid) and HPBE hydrolysis. Lip 2 also demonstrated capacity of transesterification. These applications made these enzymes attractive for industrial application.This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, nº 2013/50892-5) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, nº 406838/2013-5). This project is also part of the National Institute of Science and Technology of the Bioethanol (FAPESP, nº 2010/52322-3). MLTMP is Research Fellow of CNPq. FDAF was recipient of a FAPESP fellowship (nº 2012/14615-4). MGP and ACV are supported by CNPq.Peer reviewe